The shape and integrity of the erythrocyte is maintained by a submembraneous cytoskeleton. The major protein component of the cytoskeleton, spectrin, contains multiple association sites for other proteins and for itself. The aims of the proposed study are to: 1) identify the sites of specific functional domains within the spectrin molecule; 2) quantitate the specific regulatory roles of covalent phosphorylation; 2,3-Diphosphoglycerate, carbon dioxide, and cooperative protein binding; 3) reconstruct a credible model of the cytoskeleton in vitro; 4) relate inherited or acquired disorders of red cell shape or function to specific functional or structural aberations in the cytoskeleton. A number of methodologies will be employed. In vitro studies will use conventional purified proteins. Sensitive assays of spectrin structure and function invovle binding between radiolabeled purified components of the cytoskeleton, nondenaturing two-dimensional gel electrophoresis, and 31P nuclear magnetic resonance spectroscopy. Functional domains generated by proteolysis or by chemical cleavage can be related to the intact molecule through two-dimensional radiolabeled peptide maps and specific monoclonal antibodies. Early results suggest that these methods are sufficiently sensitive to detect single residue changes and their corresponding functional perturbations. Defects in the cytoskeleton, leading to rigid or frail erythrocytes, are thought to underlie a host of inherited and acquired disorders including hereditary spherocytosis, elliptocytosis and pyropoikilocytosis. Red cell fragility is often of concern in patients with heart valve prostheses and those with autoimmune hemolytic anemias. A clearer understanding of the factors important to red cell cytoskeletal structure and function will provide a rational approach to the control of these and similar disorders, and further our knowledge of cellular cytoskeletons.